PROJECT SUMMARY/ABSTRACT Compared to other cancers, the treatment of prostate cancer (PCa) poses a unique problem due to its high prevalence of indolent disease and high rates of treatment morbidity. PCa is the most common cancer diagnosis and the second leading cause of cancer-related mortality among US men, but 42% of US men age 50 or older that died from other causes were found to have PCa in autopsy studies. Therefore, it is essential for clinicians to accurately determine which patients will progress to lethal, metastatic PCa and which will not. Definitive diagnosis and risk stratification is based off of multi-core biopsy results. However, PCa is often multifocal with heterogeneous tumors, and the biopsy may miss areas that contain aggressive tumor. This uncertainty has led to the over-treatment of low-risk PCa. Therefore, there is an urgent clinical need for accurate pre-intervention PCa prognostic testing so that clinicians can treat high-risk patients but spare low-risk patients from the definitive treatment morbidities. To address this clinical need, the first objective of this proposal is to discover and characterize prognostic serum exosomal microRNAs (miRs) of prostatic origin. Exosomes are lipid-bilayer vesicles that are secreted from cells and function in intercellular communication but have been exploited for their potential prognostic value. These experiments capitalize on a pretreatment PCa sera cohort collected for a previously funded study. Our goal is to utilize exosomes to develop a sensitive and specific prognostic test for PCa. Previously published data suggests a prognostic role for prostate miRs, which are selectively loaded into these vesicles, so there may be a population of miRs in circulating exosomes that are specific to PCa. In addition to the prognostic potential of exosomal miRs, these vesicles have biological functions in cell-to- cell communication. Furthermore, exosomes have been implicated in tumor progression and metastasis in multiple cancers, including PCa. The most common form of PCa arises from the epithelial cells (PrE) of the glandular acini, which are surrounded by a fibromuscular stroma. The stroma functions to support epithelial growth and differentiation in the normal prostate but becomes tumor-promoting in PCa. These tumor-promoting stromal cells are termed cancer-associated fibroblasts (CAF). The second objective of this proposal is to elucidate the biological role of microRNAs in exosomes released from CAF on prostate epithelial cell aggressiveness. Human primary prostate epithelial and stromal cells derived from radical prostatectomy surgery tissue will be used as a model system to determine the changes in epithelial cell growth, proliferation, and cytokine production after treatment with exosomes from CAF. The miRs within the exosomes released from CAF will be knocked down or overexpressed to determine the mechanism of exosome action on PrE cells. This project uses patient-derived models in a translational and multi-disciplinary approach to develop a non- invasive serum exosome miR test for PCa prognosis and to determine the tumor-promoting effects of miRs released from CAF.